Project Summary/ Abstract Tuberculosis is one of the most deadly infectious diseases, responsible for the death of about 1.5 million people yearly. In 2013, an estimated 9.0 million people developed tuberculosis. Tuberculosis is caused by the bacillus Mycobacterium tuberculosis, which exploits host macrophages to survive within humans. Infected macrophages aggregate into distinctive layered structures called granulomas, the central structure of tuberculosis. At their core, tuberculous granulomas contain the vast majority of infectious bacteria within the host. These bacteria are surrounded by successive layers of immune cells including neutrophils, dendritic cells, and primarily infected and uninfected macrophages. In vivo observations in zebrafish, primates, and humans indicate that tuberculous granulomas are dynamic; however, the molecular pathways and cellular transitions underlying formation, stability, and immune cell movement within granulomas remain unclear. The long-term objective of this research is to define how immune cells harboring pathogenic mycobacteria shift their cellular identity and behave within and around granulomas. This research will provide insight into how into how infection-driven changes within immune cells alter cellular fate and the ability of host immune cells to fight disease, and may uncover adjunctive therapies to accompany the first-line antibiotics for tuberculosis treatment. Data from our laboratory suggest that macrophages entering the granuloma undergo an unusual epithelial transition and deploy molecules traditionally studied in cancer and development. We hypothesize that host immune cell identity and behavior at the granuloma draw on these pathways, which are not typically analyzed in immune cells. This objective will be achieved by modeling human tuberculosis infection using a well-established zebrafish-Mycobacterium model. The zebrafish immune system mirrors fundamental aspects of the human immune system, with both innate and adaptive arms. Mycobacterial infection in zebrafish recapitulates important aspects of human infection, and mycobacterial granulomas form over a number of days even in the sole context of innate immunity, allowing us to take advantage of the optical clarity of the larval zebrafish to directly study granuloma-associated immune cells. In this proposal transcriptional profiling, immune cell-specific knockdowns, and knockouts of developmental signaling components will be used within a zebrafish-Mycobacterium model to accomplish the following Specific Aims: 1) Determine the roles of the Shh pathway in mycobacterial pathogenesis and granuloma formation, and 2) Determine the roles of the non-canonical Wnt pathway in mycobacterial pathogenesis and granuloma epithelialization. These studies will identify an entirely new class of molecular immune cell regulators and relate them to granuloma biology in vivo in a natural host.